Heterodimeric antibodies that bind cd3 and psma

ABSTRACT

The present invention is directed to heterodimeric antibodies that bind CD3 and PSMA.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application Nos.62/264,261, filed Dec. 7, 2015 and 62/324,823, filed Apr. 19, 2016 whichare expressly incorporated by reference in their entireties.

TECHNICAL FIELD

The present disclosure relates to methods and compositions for thetreatment of cancers (e.g., prostate cancer).

BACKGROUND OF THE INVENTION

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer and autoimmune/inflammatorydisorders. Yet improvements to this class of drugs are still needed,particularly with respect to enhancing their clinical efficacy. Oneavenue being explored is the engineering of additional and novel antigenbinding sites into antibody-based drugs such that a singleimmunoglobulin molecule co-engages two different antigens. Suchnon-native or alternate antibody formats that engage two differentantigens are often referred to as bispecifics. Because the considerablediversity of the antibody variable region (Fv) makes it possible toproduce an Fv that recognizes virtually any molecule, the typicalapproach to bispecific generation is the introduction of new variableregions into the antibody.

A number of alternate antibody formats have been explored for bispecifictargeting (Chames & Baty, 2009, mAbs 1[6]:1-9; Holliger & Hudson, 2005,Nature Biotechnology 23[9]:1126-1136; Kontermann, mAbs 4(2):182 (2012),all of which are expressly incorporated herein by reference). Initially,bispecific antibodies were made by fusing two cell lines that eachproduced a single monoclonal antibody (Milstein et al., 1983, Nature305:537-540). Although the resulting hybrid hybridoma or quadroma didproduce bispecific antibodies, they were only a minor population, andextensive purification was required to isolate the desired antibody. Anengineering solution to this was the use of antibody fragments to makebispecifics. Because such fragments lack the complex quaternarystructure of a full length antibody, variable light and heavy chains canbe linked in single genetic constructs. Antibody fragments of manydifferent forms have been generated, including diabodies, single chaindiabodies, tandem scFv's, and Fab₂ bispecifics (Chames & Baty, 2009,mAbs 1[6]:1-9; Holliger & Hudson, 2005, Nature Biotechnology23[9]:1126-1136; expressly incorporated herein by reference). Whilethese formats can be expressed at high levels in bacteria and may havefavorable penetration benefits due to their small size, they clearrapidly in vivo and can present manufacturing obstacles related to theirproduction and stability. A principal cause of these drawbacks is thatantibody fragments typically lack the constant region of the antibodywith its associated functional properties, including larger size, highstability, and binding to various Fc receptors and ligands that maintainlong half-life in serum (i.e. the neonatal Fc receptor FcRn) or serve asbinding sites for purification (i.e. protein A and protein G).

More recent work has attempted to address the shortcomings offragment-based bispecifics by engineering dual binding into full lengthantibody-like formats (Wu et al., 2007, Nature Biotechnology25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al., 2009,mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000, ProteinEngineering 13[5]:361-367; U.S. Ser. No. 9/865,198; Shen et al., 2006, JBiol Chem 281[16]:10706-10714; Lu et al., 2005, J Biol Chem280[20]:19665-19672; PCT/US2005/025472; expressly incorporated herein byreference). These formats overcome some of the obstacles of the antibodyfragment bispecifics, principally because they contain an Fc region. Onesignificant drawback of these formats is that, because they build newantigen binding sites on top of the homodimeric constant chains, bindingto the new antigen is always bivalent.

For many antigens that are attractive as co-targets in a therapeuticbispecific format, the desired binding is monovalent rather thanbivalent. For many immune receptors, cellular activation is accomplishedby cross-linking of a monovalent binding interaction. The mechanism ofcross-linking is typically mediated by antibody/antigen immunecomplexes, or via effector cell to target cell engagement. For example,the low affinity Fc gamma receptors (FcγRs) such as FcγRIIa, FcγRIIb,and FcγRIIIa bind monovalently to the antibody Fc region. Monovalentbinding does not activate cells expressing these FcγRs; however, uponimmune complexation or cell-to-cell contact, receptors are cross-linkedand clustered on the cell surface, leading to activation. For receptorsresponsible for mediating cellular killing, for example FcγRIIIa onnatural killer (NK) cells, receptor cross-linking and cellularactivation occurs when the effector cell engages the target cell in ahighly avid format (Bowles & Weiner, 2005, J Immunol Methods 304:88-99,expressly incorporated by reference). Similarly, on B cells theinhibitory receptor FcγRIIb downregulates B cell activation only when itengages into an immune complex with the cell surface B-cell receptor(BCR), a mechanism that is mediated by immune complexation of solubleIgG's with the same antigen that is recognized by the BCR (Heyman 2003,Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature ReviewsImmunology 10:328-343; expressly incorporated by reference). As anotherexample, CD3 activation of T-cells occurs only when its associatedT-cell receptor (TCR) engages antigen-loaded MHC on antigen presentingcells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of CD3using an anti-CD3 antibody elicits a cytokine storm and toxicity(Penuche et al., 2009, J Immunol 183[2]:953-61; Chatenoud & Bluestone,2007, Nature Reviews Immunology 7:622-632; expressly incorporated byreference). Thus for practical clinical use, the preferred mode of CD3co-engagement for redirected killing of targets cells is monovalentbinding that results in activation only upon engagement with theco-engaged target.

Thus while bispecifics generated from antibody fragments sufferbiophysical and pharmacokinetic hurdles, a drawback of those built withfull length antibody-like formats is that they engage co-target antigensmultivalently in the absence of the primary target antigen, leading tononspecific activation and potentially toxicity. The present inventionsolves this problem by introducing novel bispecific antibodies directedto CD3 and prostate specific membrane antigen (PSMA).

BRIEF SUMMARY

Provided herein are heterodimeric antibodies that bind to CD3 andprostrate specific membrane antigen (PSMA), a transmembrane protein thatis considerably overexpressed on most prostate cancer cells. Suchantibodies are useful for the treatment of cancers (e.g., prostratecancer), for example, by facilitating the targeting CD3+ T cells to PSMAexpressing tumor cells.

In one aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer that includes SEQ ID NO: 64; b) a secondmonomer that includes SEQ ID NO: 65; and c) a light chain comprising SEQID NO: 66. In a further aspect, provided herein is a nucleic acidcomposition that includes: a) a first nucleic acid encoding a firstmonomer that includes SEQ ID NO: 64; b) a second nucleic acid encoding asecond monomer that includes SEQ ID NO: 65; and c) a third nucleic acidencoding a light chain that includes SEQ ID NO: 66. In another aspect,provided herein is an expression vector composition that includes: a) afirst expression vector that includes a first nucleic acid encoding afirst monomer that includes SEQ ID NO: 64; b) a second expression vectorthat includes a second nucleic acid encoding a second monomer thatincludes SEQ ID NO: 65; and c) a third expression vector that includes athird nucleic acid encoding a light chain that includes SEQ ID NO: 66.

In another aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer that includes SEQ ID NO: 118; b) a secondmonomer that includes SEQ ID NO: 119; and c) a light chain that includesSEQ ID NO: 120. In a further aspect, provided herein is a nucleic acidcomposition that includes: a) a first nucleic acid encoding a firstmonomer that includes SEQ ID NO: 118; b) a second nucleic acid encodinga second monomer that includes SEQ ID NO: 119; and c) a third nucleicacid encoding a light chain that includes SEQ ID NO: 120. In anotheraspect, provided herein is an expression vector composition thatincludes: a) a first expression vector that includes a first nucleicacid encoding a first monomer that includes SEQ ID NO: 118; b) a secondexpression vector that includes a second nucleic acid encoding a secondmonomer that includes SEQ ID NO: 119; and c) a third expression vectorcomprising a nucleic acid that includes a third nucleic acid encoding alight chain that includes g SEQ ID NO: 120.

In one aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer that includes SEQ ID NO: 121; b) a secondmonomer that includes SEQ ID NO: 122; and c) a light chain that includesSEQ ID NO: 123. In another aspect, provided herein is a nucleic acidcomposition that includes: a) a first nucleic acid encoding a firstmonomer that includes SEQ ID NO: 121; b) a second nucleic acid encodinga second monomer that includes SEQ ID NO: 122; and c) a third nucleicacid encoding a light chain that includes SEQ ID NO: 123. In a furtheraspect, provided herein is an expression vector composition thatincludes: a) a first expression vector that includes a first nucleicacid encoding a first monomer that includes SEQ ID NO: 121; b) a secondexpression vector that includes a second nucleic acid encoding a secondmonomer that includes SEQ ID NO: 122; and c) a third expression vectorthat includes a nucleic acid encoding a third nucleic acid encoding alight chain that includes SEQ ID NO: 123.

In one aspect, provided herein is a heterodimeric antibody thatincludes: a) a first monomer that includes SEQ ID NO: 124; b) a secondmonomer that includes SEQ ID NO: 125; and c) a light chain that includesSEQ ID NO: 126. In another aspect, provided herein is a nucleic acidcomposition that includes: a) a first nucleic acid encoding a firstmonomer that includes SEQ ID NO: 124; b) a second nucleic acid encodinga second monomer that includes SEQ ID NO: 125; and c) a third nucleicacid encoding a light chain that includes SEQ ID NO: 126. In yet anotheraspect, provided herein is an expression vector composition thatincludes: a) a first expression vector that includes a first nucleicacid encoding a first monomer that includes SEQ ID NO: 124; b) a secondexpression vector that includes a second nucleic acid encoding a secondmonomer that includes SEQ ID NO: 125; and c) a third expression vectorthat includes a nucleic acid encoding a third nucleic acid encoding alight chain that includes SEQ ID NO: 126.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a light chain. The first monomerincludes: i) a first Fc domain; ii) an anti-CD3 scFv that includes ascFv variable light domain, and an scFv linker and a scFv variable heavydomain; where the scFv is covalently attached to the N-terminus of theFc domain using a domain linker. The second monomer includes a heavychain that includes: i) a heavy variable domain; and ii) a heavyconstant domain that includes a second Fc domain. The light chainincludes a variable light domain and a constant light domain. The heavyvariable domain and the variable light domain bind PSMA.

In another aspect, provided herein is a heterodimeric antibody thatincludes a first monomer, a second monomer and a light chain. The firstmonomer includes: i) a first Fc domain; and ii) an anti-CD3 scFv thatincludes a scFv variable light domain, an scFv linker and a scFvvariable heavy domain; where the scFv is covalently attached to theN-terminus of the Fc domain using a domain linker. The second monomerincludes a heavy chain that includes i) a heavy variable domain; and ii)a heavy constant domain that includes a second Fc domain. The lightchain includes a variable light domain and a constant light domain. ThescFv variable light domain includes: the vlCDRs of CD3 L1.47 (a vlCDR1having SEQ ID NO: 6, a vlCDR2 having SEQ ID NO: 7, and a vlCDR3 havingSEQ ID NO: 8), the scFv variable heavy domain includes the vhCDRs of CD3H1.30 (a vhCDR1 having SEQ ID NO: 2, a vhCDR2 having SEQ ID NO: 3, and avhCDR3 having SEQ ID NO: 4), and the heavy variable domain and thevariable light domain bind PSMA.

In another aspect, provided herein is a heterodimeric antibody thatincludes a first monomer, a second monomer and a light chain. The firstmonomer includes: i) a first Fc domain; and ii) an anti-CD3 scFv thatincludes a scFv variable light domain, an scFv linker and a scFvvariable heavy domain; where the scFv is covalently attached to theN-terminus of the Fc domain using a domain linker. The second monomerincludes a heavy chain that includes: i) a heavy variable domain; andii) a heavy constant domain that includes a second Fc domain. The lightchain includes a variable light domain and a constant light domain. ThescFv variable light domain includes: the vlCDRs of CD3 L1.47 (a vlCDR1having SEQ ID NO: 15, a vlCDR2 having SEQ ID NO: 16, and a vlCDR3 havingSEQ ID NO: 17), the scFv variable heavy domain includes the vhCDRs ofCD3 H1.32 (a vhCDR1 having SEQ ID NO: 11, a vhCDR2 having SEQ ID NO: 12,and a vhCDR3 having SEQ ID NO: 13), and the heavy variable domain andthe variable light domain bind PSMA.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a light chain. The first monomerincludes: i) a first Fc domain; and ii) an anti-CD3 scFv that includes ascFv variable light domain, an scFv linker and a scFv variable heavydomain; where the scFv is covalently attached to the N-terminus of theFc domain using a domain linker. The second monomer includes a heavychain that includes: i) a heavy variable domain; and ii) a heavyconstant domain that includes a second Fc domain. The light chainincludes a variable light domain and a constant light domain. The scFvvariable light domain includes the vlCDRs of CD3 L1.47 (a vlCDR1 havingSEQ ID NO: 24, a vlCDR2 having SEQ ID NO: 25, and a vlCDR3 having SEQ IDNO: 26), the scFv variable heavy domain includes the vhCDRs of CD3 H1.89(a vhCDR1 having SEQ ID NO: 20, a vhCDR2 having SEQ ID NO: 21, and avhCDR3 having SEQ ID NO: 22), and the heavy variable domain and thevariable light domain bind PSMA.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a light chain. The first monomerincludes: i) a first Fc domain; and ii) an anti-CD3 scFv that includes ascFv variable light domain, an scFv linker and a scFv variable heavydomain; where the scFv is covalently attached to the N-terminus of theFc domain using a domain linker. The second monomer includes a heavychain that includes: i) a heavy variable domain; and ii) a heavyconstant domain that includes a second Fc domain. The light chainincludes a variable light domain and a constant light domain. The scFvvariable light domain includes: the vlCDRs of CD3 L1.47 (a vlCDR1 havingSEQ ID NO: 33, a vlCDR2 having SEQ ID NO: 34, and a vlCDR3 having SEQ IDNO: 35), the scFv variable heavy domain includes the vhCDRs of CD3 H1.90(a vhCDR1 having SEQ ID NO: 29, a vhCDR2 having SEQ ID NO: 30, and avhCDR3 having SEQ ID NO: 31), and the heavy variable domain and thevariable light domain bind PSMA.

In another aspect, provided herein is a heterodimeric antibody thatincludes a first monomer, a second monomer and a light chain. The firstmonomer includes: i) a first Fc domain; and ii) an anti-CD3 scFv thatincludes a scFv variable light domain, an scFv linker and a scFvvariable heavy domain; where the scFv is covalently attached to theN-terminus of the Fc domain using a domain linker. The second monomerincludes a heavy chain that includes: i) a heavy variable domain; andii) a heavy constant domain that includes a second Fc domain. The lightchain includes a variable light domain and a constant light domain. ThescFv variable light domain includes: the vlCDRs of CD3 L1.47 (a vlCDR1having SEQ ID NO: 42, a vlCDR2 having SEQ ID NO: 43, and a vlCDR3 havingSEQ ID NO: 44), the scFv variable heavy domain includes the vhCDRs ofCD3 H1.33 (a vhCDR1 having SEQ ID NO: 38, a vhCDR2 having SEQ ID NO: 39,and a vhCDR3 having SEQ ID NO: 40), and the heavy variable domain andthe variable light domain bind PSMA.

In another aspect, provided herein is a heterodimeric antibody thatincludes a first monomer, a second monomer and a light chain. The firstmonomer includes: i) a first Fc domain; and ii) an anti-CD3 scFv thatincludes a scFv variable light domain, an scFv linker and a scFvvariable heavy domain; where the scFv is covalently attached to theN-terminus of the Fc domain using a domain linker. The second monomerincludes a heavy chain that includes: i) a heavy variable domain; andii) a heavy constant domain that includes a second Fc domain. The lightchain includes a variable light domain and a constant light domain. ThescFv variable light domain includes: the vlCDRs of CD3 L1.47 (a vlCDR1having SEQ ID NO: 51, a vlCDR2 having SEQ ID NO: 52, and a vlCDR3 havingSEQ ID NO: 53), the scFv variable heavy domain includes the vhCDRs ofCD3 H1.31 (a vhCDR1 having SEQ ID NO: 47, a vhCDR2 having SEQ ID NO: 48,and a vhCDR3 having SEQ ID NO: 49), and the heavy variable domain andthe variable light domain bind PSMA.

In one embodiment of the antibodies described above, the heavy variabledomain includes the vhCDRs of anti-PSMA J591 H1_L1 (a vhCDR1 having SEQID NO: 111, a vhCDR2 having SEQ ID NO: 112, and a vhCDR3 having SEQ IDNO: 113) and the variable light domain includes the vlCDRs of anti-PSMAJ591 H1_L1 (a vlCDR1 having SEQ ID NO: 107, a vlCDR2 having SEQ ID NO:108, and a vlCDR3 having SEQ ID NO: 109). In an exemplary embodiment,the heavy variable domain includes the variable heavy domain ofanti-PSMA J591 H1_L1 (SEQ ID NO: 110) and the variable light domainincludes the variable light domain of anti-PSMA J591 H1_L1 (SEQ ID NO:106).

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a light chain. The first monomerincludes: i) a first Fc domain; and ii) an anti-CD3 scFv that includes ascFv variable light domain, an scFv linker and a scFv variable heavydomain; where the scFv is covalently attached to the N-terminus of theFc domain using a domain linker. The second monomer includes a heavychain that includes: i) a heavy variable domain; and ii) a heavyconstant domain that includes a second Fc domain. The light chainincludes a variable light domain and a constant light domain. The heavyvariable domain includes the vhCDRs of anti-PSMA J591 H1_L1 (a vhCDR1having SEQ ID NO: 111, a vhCDR2 having SEQ ID NO: 112, and a vhCDR3having SEQ ID NO: 113) and the variable light domain includes the vlCDRsof anti-PSMA J591 H1_L1 (a vlCDR1 having SEQ ID NO: 107, a vlCDR2 havingSEQ ID NO: 108, and a vlCDR3 having SEQ ID NO: 109). The heavy variabledomain and the variable light domain bind PSMA. In some embodiments, theheavy variable domain includes the variable heavy domain of anti-PSMAJ591 H1_L1 (SEQ ID NO: 110) and the variable light domain includes thevariable light domain of anti-PSMA J591 H1_L1 (SEQ ID NO: 106).

In some embodiments of the antibodies described above, the first Fcdomain and the second Fc domain comprise a set of variants selected fromthe group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L andK370S:S364K/E357Q.

In some embodiments of the antibodies described above, the scFv linkeris a charged linker.

In some embodiments of the antibodies described above, the heavy chainconstant domain includes the amino acid substitutionsN208D/Q295E/N384D/Q418E/N421D.

In some embodiments of the antibodies described above, the first andsecond Fc domains include the amino acid substitutionsE233P/L234V/L235A/G236del/S267K.

In another aspect, provided herein is a heterodimeric antibody thatincludes a first monomer, a second monomer and a common light chain. Thefirst monomer includes: i) a first heavy chain that includes: 1) a firstvariable heavy domain; 2) a first constant heavy chain that includes afirst Fc domain; and 3) a scFv that includes a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain; where the scFvis covalently attached to the C-terminus of the Fc domain using a domainlinker. The second monomer includes a second heavy chain that includes asecond variable heavy domain and a second constant heavy chain thatincludes a second Fc domain. The common light chain includes a variablelight domain and a constant light domain. The first and the second Fcdomains have a set of amino acid substitutions selected from the groupconsisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L andK370S:S364K/E357Q. The first variable heavy domain and the variablelight domain bind human PSMA (SEQ ID NO:131), the second variable heavydomain and the variable light domain bind human PSMA, and the scFv bindshuman CD3.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a common light chain. The firstmonomer includes i) a first heavy chain that includes: 1) a firstvariable heavy domain; 2) a first constant heavy domain that includes afirst Fc domain; and 3) a first variable light domain, where the firstvariable light domain is covalently attached to the C-terminus of thefirst Fc domain using a domain linker. The second monomer includes: i) asecond variable heavy domain; ii) a second constant heavy domain thatincludes a second Fc domain; and iii) a third variable heavy domain,where the third variable heavy domain is covalently attached to theC-terminus of the second Fc domain using a domain linker. The commonlight chain includes a second variable light domain and a constant lightdomain. The first and the second Fc domains have a set of amino acidsubstitutions selected from the group consisting ofS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and K370S:S364K/E357Q.The first variable heavy domain and the second variable light domainbind PSMA, the second variable heavy domain and the second variablelight domain bind the PSMA, and the first variable light domain and thethird variable heavy domain bind a CD3.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a common light chain. The firstmonomer includes i) a first heavy chain that includes i) a first heavychain that includes: 1) a first variable heavy domain; 2) a firstconstant heavy chain that includes a first CH1 domain and a first Fcdomain; and 3) a scFv that includes a scFv variable light domain, anscFv linker and a scFv variable heavy domain; where the scFv iscovalently attached between the C-terminus of the CH1 domain and theN-terminus of the first Fc domain using domain linkers The secondmonomer includes a second heavy chain that includes a second variableheavy domain and a second constant heavy chain that includes a second Fcdomain. The common light chain includes a variable light domain and aconstant light domain. The first and the second Fc domain have a set ofamino acid substitutions selected from the group consisting ofS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and K370S:S364K/E357Q.The first variable heavy domain and the variable light domain bind PSMA,the second variable heavy domain and the variable light domain bindPSMA, and the scFv binds CD3.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a common light chain. The firstmonomer includes i) a first heavy chain that includes i) a firstvariable heavy domain; ii) a first constant heavy domain that includes afirst Fc domain; and iii) a first variable light domain, where the firstvariable light domain is covalently attached between the C-terminus ofthe CH1 domain of the first constant heavy domain and the N-terminus ofthe first Fc domain using domain linkers. The second monomer includes:i) a second variable heavy domain; ii) a second constant heavy domainthat includes a second Fc domain; and iii) a third variable heavydomain, where the third variable heavy domain is covalently attachedbetween the C-terminus of the CH1 domain of the second constant heavydomain and the N-terminus of the second Fc domain using domain linker.The common light chain includes a second variable light domain and aconstant light domain. The first and the second Fc domains have a set ofamino acid substitutions selected from the group consisting ofS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and K370S:S364K/E357Q.The first variable heavy domain and the second variable light domainbind PSMA, the second variable heavy domain and the second variablelight domain bind PSMA, and the first variable light domain and thethird variable heavy domain binds CD3.

In one aspect, provided herein is a heterodimeric antibody that includesa first monomer, a second monomer and a light chain. The first monomerincludes i) a first heavy chain that includes i) a first heavy chainthat includes: 1) a first variable heavy domain; 2) a first constantheavy chain that includes a first CH1 domain and a first Fc domain; and3) a scFv that includes a scFv variable light domain, an scFv linker anda scFv variable heavy domain; where the scFv is covalently attachedbetween the C-terminus of the CH1 domain and the N-terminus of the firstFc domain using domain linkers The second monomer includes a second Fcdomain. The light chain includes a variable light domain and a constantlight domain. The first and the second Fc domain have a set of aminoacid substitutions selected from the group consisting ofS364K/E357Q:L368D/K370S; L368D/K370S:S 364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S 364K/E357L and K370S:S364K/E357Q.The first variable heavy domain and the variable light domain bind afirst antigen, the scFv binds a second antigen, and one of the first andsecond antigens is PSMA and the other is CD3.

In another aspect, provided herein is a nucleic acid composition thatencodes any of the subject heterodimeric antibodies described herein. Insome embodiments, the nucleic acid composition includes a) a firstnucleic acid encoding the first monomer; b) a second nucleic acidencoding the second monomer; and c) a third nucleic acid encoding thelight chain.

In another aspect, provided herein is a heterodimeric antibody selectedfrom the group consisting of: XENP14484, XENP16873, XENP16874 andXENP19722.

In another aspect, provided herein is an expression vector compositionthat includes any one of the nucleic acid compositions described herein.In another aspect, provided herein is a host cell that includes any oneof the nucleic acid compositions or expression vector compositionsdescribed herein. In yet another aspect, provided herein is a method ofmaking a subject heterodimeric antibody described herein that includesculturing a host cell described herein under conditions where theantibody is expressed, and recovering the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict several formats of the present invention. Twoforms of the “bottle opener” format are depicted, one with the anti-CD3antigen binding domain comprising a scFv and the anti-PSMA antigenbinding domain comprising a Fab, and one with these reversed. ThemAb-Fv, mAb-scFv, Central-scFv and Central-Fv, common light chain, andbispecific IgG formats are all shown. While each of the structures inthese figures depict the anti-PSMA and anti-CD3 domains as an scFv or aFv, the anti-PSMA and anti-CD3 domains of each of these structures canalso be “reversed”. For example, it is also contemplated that themAb-scFv depicted in FIG. 1A can have an anti-PSMA scFv and an anti-CD3Fv. In addition, “one-armed” formats, where one monomer just comprisesan Fc domain, are shown, including a one arm Central-scFv and a one armCentral-Fv. A dual scFv format is also shown.

FIG. 2 depicts the sequences of the “High CD3” anti-CD3_H1.30_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 3 depicts the sequences of the “High-Int #1” Anti-CD3_H1.32_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 4 depicts the sequences of the “High-Int #2” Anti-CD3_H1.89_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 5 depicts the sequences of the “High-Int #3” Anti-CD3_H1.90_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker, asneeded.

FIG. 6 depicts the sequences of the “Int” Anti-CD3_H1.33_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker (e.g., anycharged linker in FIG. 14), as needed.

FIG. 7 depicts the sequences of the “Low” Anti-CD3_H1.31_L1.47construct, including the variable heavy and light domains (CDRsunderlined), as well as the individual vl and vhCDRs, as well as an scFvconstruct with a charged linker (double underlined). As is true of allthe sequences depicted in the Figures, this charged linker may bereplaced by an uncharged linker or a different charged linker (e.g., anycharged linker in FIG. 14), as needed.

FIG. 8 depicts the sequences of the variable heavy and variable lightdomains of XENP14484, including the variable heavy and light domains(CDRs underlined), as well as the individual vl and vhCDRs, as well asan scFv construct with a charged linker (double underlined). However, aswill be appreciated by those in the art, the charged linker in the caseof the PSMA embodiment, the positively charged linker can also bereplaced by a negatively charged one as shown in FIG. 14, or anuncharged traditional GS linker.

FIG. 9 depicts the sequences of the XENP14484.

FIGS. 10A-10E depict useful pairs of heterodimerization variant sets(including skew and pI variants). On FIG. 10E, there are variants forwhich there are no corresponding “monomer 2” variants; these are pIvariants which can be used alone on either monomer, or included on theFab side of a bottle opener, for example, and an appropriate chargedscFv linker can be used on the second monomer that utilizes a scFv asthe second antigen binding domain. Suitable charged linkers are shown inFIG. 14.

FIG. 11 depict a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theinvention (and other variant types as well, as outlined herein).

FIG. 12 depict useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants).

FIG. 13 show two particularly useful embodiments of the invention.

FIGS. 14A-14B depict a number of charged scFv linkers that find use inincreasing or decreasing the pI of heterodimeric antibodies that utilizeone or more scFv as a component. The (+H) positive linker findsparticular use herein, particularly with anti-CD3 vl and vh sequencesshown herein. A single prior art scFv linker with a single charge isreferenced as “Whitlow”, from Whitlow et al., Protein Engineering6(8):989-995 (1993). It should be noted that this linker was used forreducing aggregation and enhancing proteolytic stability in scFvs.

FIG. 15 depicts a list of engineered heterodimer-skewing Fc variantswith heterodimer yields (determined by HPLC-CIEX) and thermalstabilities (determined by DSC). Not determined thermal stability isdenoted by “n.d.”.

FIG. 16. Test articles are anti-PSMA or anti-RSV x anti-CD3 bispecifics.Top panel: Cell surface binding to 100k LNCaP cells. Detection was by aFITC-labeled anti-human IgG Fc secondary antibody. Bottom panel:Redirected T cell cytotoxicity assay, 24 h incubation, 10k LNCaP cells,400k purified T cells. Detection was by LDH.

FIGS. 17A and 17B include graphs showing that T cell response andcytokine release syndrome (CRS) is dependent on the engagement of targetcells in monkeys. Cynomolgus monkeys (n=3) were given a single dose ofeither XENP14484 (anti-PSMA x anti-CD3, 30 μg/kg) or XENP13245 (anti-RSVx CD3, 3 mg/kg). FIG. 17A CD4′ T cell margination (top panel) andactivation by CD69 MFI (middle panel) were examined by flow cytometry.IL-6 serum levels were examined by ELISA (bottom panel). FIG. 17B CD8′ Tcell margination (top panel) and activation by CD69 MFI (middle panel)were examined by flow cytometry. TNF serum levels were examined by ELISA(bottom panel).

FIG. 18 shows a matrix of possible combinations for embodiments of theinvention. An “A” means that the CDRs of the referenced anti-CD3sequences can be combined with the CDRs of the anti-PSMA construct onthe left hand side. That is, for example for the top left hand cell, thevhCDRs from the variable heavy chain anti-CD3 H1.30 sequence and thevlCDRs from the variable light chain of anti-CD3 L1.47 sequence can becombined with the vhCDRs from the anti-PSMA J591 H1_L1 sequence and thevlCDRs from the J591 H1_L1 sequence (see FIG. 20). A “B” means that theCDRs from the anti-CD3 constructs can be combined with the variableheavy and light domains from the anti-PSMA construct. That is, forexample for the top left hand cell, the vhCDRs from the variable heavychain anti-CD3 H1.30 sequence and the vlCDRs from the variable lightchain of anti-CD3 L1.47 sequence can be combined with the variable heavydomain J591 H1_L1 sequence and the J591 H1_L1 sequence. A “C” isreversed, such that the variable heavy domain and variable light domainfrom the CD3 sequences are used with the CDRs of the PSMA sequences. A“D” is where both the variable heavy and variable light chains from eachare combined. An “E” is where the scFv of the anti-CD3 is used with theCDRs of the PSMA antigen binding domain construct. An “F” is where thescFv of the CD3 is used with the variable heavy and variable lightdomains of the PSMA antigen binding domain. A “G” is where the CDRs ofthe anti-CD3 constructs are used with the scFv of the PSMA constuct. An“H” is where the variable heavy domain and variable light domain of theanti-CD3 is used with the scFv of the anti-PSMA.

FIG. 19 depicts amino acid sequences for various prostate-specificmembrane antigens (PSMAs), including full human PSMA, human PSMAextracellular domain, full Macaca fascicularis PSMA, and Macacafascicularis PSMA extracellular domain.

FIG. 20 depicts the amino acid sequences for the variable heavy (VH) andvariable light (VL) domains of unhumanized anti-PSMA antibody J591 andhumanized anti-PSMA antibody J591 H1_L1. Also depicted are thecomplementary determining regions of the variable heavy (vhCDR1-3) andvariable light (vlCDR1-3) domains included in these variable heavy andvariable light domains. In some embodiments, the anti-PSMA CDRs and VHand VLs can be used in monospecific and heterodimer antibodies (e.g.,anti-PSMA x anti-CD3) antibodies described herein. In some embodiments,wherein the heterodimeric antibody further includes an anti-CD3 bindingdomain, the anti-CD3 binding domain can include any of the VH, VL orCDRs depicted in FIGS. 5-7.

FIGS. 21A-21E depict the amino acid sequences of exemplary anti-PSMA xanti-CD3 antibodies described herein, including XENP14484 (FIG. 21A),XENP16873 (FIG. 21B), XENP16874 (FIG. 21C), and XENP19722 (FIG. 21D).FIG. 22E depicts the amino acid sequence of an exemplary bottle openerscaffold. Such a scaffold can be combined with the various variableheavy domains, variable light domains, and scFvs described herein.

FIG. 22 depicts a graph showing the PSMA affinity (Kd) determination ofanti-PSMA x anti-CD3 antibodies XENP14484 and XENP19722.

FIG. 23 are graphs showing the binding of exemplary anti-PSMA x anti-CD3antibodies XENP14484 and XENP 19722 to LNCaP prostate cancer cells(LNCaP)(top) and the ability of such exemplary antibodies to mediateredirected T cell cytotoxicity (RTCC) of a LNCaP prostate cancer cellline (bottom). Top panel: Cell surface binding to 100k LNCaP cells.Detection was by a FITC-labeled anti-human IgG Fc secondary antibody.Bottom panel: Redirected T cell cytotoxicity assay, overnightincubation, 10k LNCaP cells, 100k purified T cells from human PBMC.Detection was by LDH.

FIG. 24 are graphs showing that an anti-PSMA x anti-CD3 bispecificantibody with reduced CD3 affinity (XENP19722) exhibits similar T cellactivation (top, middle) but lower CRS in monkeys as compared to an ananti-PSMA x anti-CD3 bispecific antibody with high CD3 affinity(XENP14484)(bottom).

FIGS. 25A-25D shows the sequences of several useful bottle opener formatbackbones, without the Fv sequences (e.g. the scFv and the vh and vl forthe Fab side). As will be appreciated by those in the art and outlinedbelow, these sequences can be used with any vh and vl pairs outlinedherein, with one monomer including a scFv (optionally including acharged scFv linker) and the other monomer including the Fab sequences(e.g. a vh attached to the “Fab side heavy chain” and a vl attached tothe “constant light chain”). The scFv can be anti-CD3 or anti-TTA, withthe Fab being the other. That is, any anti-CD3 and anti-PSMA Fvsequences outlined herein, including the CDRs of such sequences, for canbe incorporated into these FIG. 25 backbones in any combination.

It should be noted that these bottle opener backbones find use in theCentral-scFv format of FIG. 1B, where an additional, second Fab (vh-CH1and vl-constant light) with the same antigen binding as the first Fab isadded to the N-terminus of the scFv on the “bottle opener side”.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding are those shown in FIG. 12.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233# or E233( ) designatesa deletion of glutamic acid at position 233. Additionally, EDA233- orEDA233# designates a deletion of the sequence GluAspAla that begins atposition 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”, for example aIgG1/2 hybrid as previously disclosed. The protein variant sequenceherein will preferably possess at least about 80% identity with a parentprotein sequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Variant protein can referto the variant protein itself, compositions comprising the proteinvariant, or the DNA sequence that encodes it. Accordingly, by “antibodyvariant” or “variant antibody” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification, “IgG variant” or “variant IgG” as used herein is meant anantibody that differs from a parent IgG (again, in many cases, from ahuman IgG sequence) by virtue of at least one amino acid modification,and “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain. The Fcvariants of the present invention are defined according to the aminoacid modifications that compose them. Thus, for example, N434S or 434Sis an Fc variant with the substitution serine at position 434 relativeto the parent Fc polypeptide, wherein the numbering is according to theEU index. Likewise, M428L/N434S defines an Fc variant with thesubstitutions M428L and N434S relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 428L/434S. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 428L/434S is the same Fc variant as M428L/N434S, andso on. For all positions discussed in the present invention that relateto antibodies, unless otherwise noted, amino acid position numbering isaccording to the EU index. The EU index or EU index as in Kabat or EUnumbering scheme refers to the numbering of the EU antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporatedby reference.) The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand, in some cases, synthetic amino acids. Examples include U.S. Pat.No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of theAmerican Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz,(2002), Chem Bio Chem 11:1135-1137; J. W. Chin, et al., (2002), PICASUnited States of America 99:11020-11024; and, L. Wang, & P. G. Schultz,(2002), Chem. 1-10, all entirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody. As will beappreciated by those in the art, these generally are made up of twochains.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcqammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et at, 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants are used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life as outlined herein, such as428L/434S.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain and in some cases, part ofthe hinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3)and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR receptors or to the FcRn receptor.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof an antibody.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein. In some cases, onemonomer of the heterodimeric antibody comprises an antibody heavy chain(either including an scFv or further including a light chain) and theother monomer is a Fc fusion, comprising a variant Fc domain and aligand. In some embodiments, these “half antibody-half fusion proteins”are referred to as “Fusionbodies”.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “strandedness” in the context of the monomers of the heterodimericantibodies of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities. Similarly,for “skew” variants that come in pairs of a set as more fully outlinedbelow, the skilled artisan will consider pI in deciding into whichstrand or monomer that incorporates one set of the pair will go, suchthat pI separation is maximized using the pI of the skews as well.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10-4 M, at least about 10-5 M, at least about10-6 M, at least about 10-7 M, at least about 10-8 M, at least about10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, atleast about 10-12 M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

II. Overview

Bispecific antibodies that co-engage CD3 and a tumor antigen target havebeen designed and used to redirect T cells to attack and lyse targetedtumor cells. Examples include the BiTE and DART formats, whichmonovalently engage CD3 and a tumor antigen. While the CD3-targetingapproach has shown considerable promise, a common side effect of suchtherapies is the associated production of cytokines, often leading totoxic cytokine release syndrome. Because the anti-CD3 binding domain ofthe bispecific antibody engages all T cells, the high cytokine-producingCD4 T cell subset is recruited. Moreover, the CD4 T cell subset includesregulatory T cells, whose recruitment and expansion can potentially leadto immune suppression and have a negative impact on long-term tumorsuppression. In addition, these formats do not contain Fc domains andshow very short serum half-lives in patients.

While the CD3-targeting approach has shown considerable promise, acommon side effect of such therapies is the associated production ofcytokines, often leading to toxic cytokine release syndrome. Because theanti-CD3 binding domain of the bispecific antibody engages all T cells,the high cytokine-producing CD4 T cell subset is recruited. Moreover,the CD4 T cell subset includes regulatory T cells, whose recruitment andexpansion can potentially lead to immune suppression and have a negativeimpact on long-term tumor suppression. One such possible way to reducecytokine production and possibly reduce the activation of CD4 T cells isby reducing the affinity of the anti-CD3 domain for CD3.

Accordingly, in some embodiments the present invention provides antibodyconstructs comprising anti-CD3 antigen binding domains in combinationwith antigen binding domains that bind prostate specific membraneantigen (PSMA).

Furthermore, in some embodiments, the anti-CD3 antigen binding domainsare “strong” or “high affinity” binders to CD3 (e.g. one example areheavy and light variable domains depicted as H1.30_L1.47 (optionallyincluding a charged linker as appropriate)) and also bind to PSMA. Inother embodiments, the present invention provides antibody constructscomprising anti-CD3 antigen binding domains that are “lite” or “loweraffinity” binders to CD3. Additional embodiments provides antibodyconstructs comprising anti-CD3 antigen binding domains that haveintermediate or “medium” affinity to CD3 that also bind to PSMA.Affinity is generally measured using a Biacore assay.

It should be appreciated that the “high, medium, low” anti-CD3 sequencesof the present invention can be used in a variety of heterodimerizationformats. While the majority of the disclosure herein uses the “bottleopener” format of heterodimers, these variable heavy and lightsequences, as well as the scFv sequences (and Fab sequences comprisingthese variable heavy and light sequences) can be used in other formats,such as those depicted in FIG. 2 of WO Publication No. 2014/145806, theFigures, formats and legend of which is expressly incorporated herein byreference.

Accordingly, the present invention provides heterodimeric antibodiesthat bind to two different antigens, e.g the antibodies are“bispecific”, in that they bind two different target antigens, e.g. CD3and PSMA in the present invention. These heterodimeric antibodies canbind these target antigens either monovalently (e.g. there is a singleantigen binding domain such as a variable heavy and variable lightdomain pair) or bivalently (there are two antigen binding domains thateach independently bind the antigen). The heterodimeric antibodies ofthe invention are based on the use different monomers which containamino acid substitutions that “skew” formation of heterodimers overhomodimers, as is more fully outlined below, coupled with “pI variants”that allow simple purification of the heterodimers away from thehomodimers, as is similarly outlined below. For the heterodimericbispecific antibodies of the invention, the present invention generallyrelies on the use of engineered or variant Fc domains that canself-assemble in production cells to produce heterodimeric proteins, andmethods to generate and purify such heterodimeric proteins.

III. Antibodies

The present invention relates to the generation of bispecific antibodiesthat bind CD3 and PSMA, generally therapeutic antibodies. As isdiscussed below, the term “antibody” is used generally. Antibodies thatfind use in the present invention can take on a number of formats asdescribed herein, including traditional antibodies as well as antibodyderivatives, fragments and mimetics, described herein.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. It should be noted that IgG1has different allotypes with polymorphisms at 356 (D or E) and 358 (L orM). The sequences depicted herein use the 356D/358M allotype, howeverthe other allotype is included herein. That is, any sequence inclusiveof an IgG1 Fc domain included herein can have 356E/358L replacing the356D/358M allotype.

In addition, many of the sequences herein have at least one thecysteines at position 220 replaced by a serine; generally this is the onthe “scFv monomer” side for most of the sequences depicted herein,although it can also be on the “Fab monomer” side, or both, to reducedisulfide formation. Specifically included within the sequences hereinare one or both of these cysteines replaced (C220S).

Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. It should be understood that therapeuticantibodies can also comprise hybrids of isotypes and/or subclasses. Forexample, as shown in US Publication 2009/0163699, incorporated byreference, the present invention covers pI engineering of IgG1/G2hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated CDRs. Accordingly, the disclosure of each variable heavyregion is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2 and vhCDR3)and the disclosure of each variable light region is a disclosure of thevlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g, Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

It should be noted that the sequences depicted herein start at the CH1region, position 118; the variable regions are not included except asnoted. For example, the first amino acid of SEQ ID NO: 2, whiledesignated as position“1” in the sequence listing, corresponds toposition 118 of the CH1 region, according to EU numbering.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cx).

Another region of interest for additional substitutions, outlined below,is the Fc region.

Thus, the present invention provides different antibody domains. Asdescribed herein and known in the art, the heterodimeric antibodies ofthe invention comprise different domains within the heavy and lightchains, which can be overlapping as well. These domains include, but arenot limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc domainor CH1-hinge-CH2-CH3), the variable heavy domain, the variable lightdomain, the light constant domain, FAb domains and scFv domains.

Thus, the “Fc domain” includes the —CH2-CH3 domain, and optionally ahinge domain. In the embodiments herein, when a scFv is attached to anFc domain, it is the C-terminus of the scFv construct that is attachedto the hinge of the Fc domain; for example, it is generally attached tothe sequence EPKS which is the beginning of the hinge. The heavy chaincomprises a variable heavy domain and a constant domain, which includesa CH1-optional hinge-Fc domain comprising a CH2-CH3. The light chaincomprises a variable light chain and the light constant domain. A scFvcomprises a variable heavy chain, an scFv linker, and a variable lightdomain. In most of the constructs and sequences outlined herein,C-terminus of the variable light chain is attached to the N-terminus ofthe scFv linker, the C-terminus of which is attached to the N-terminusof a variable heavy chain (N-vh-linker-vl-C) although that can beswitched (N-vl-linker-vh-C).

Some embodiments of the invention comprise at least one scFv domain,which, while not naturally occurring, generally includes a variableheavy domain and a variable light domain, linked together by a scFvlinker. As shown herein, there are a number of suitable scFv linkersthat can be used, including traditional peptide bonds, generated byrecombinant techniques.

The linker peptide may predominantly include the following amino acidresidues: Gly, Ser, Ala, or Thr. The linker peptide should have a lengththat is adequate to link two molecules in such a way that they assumethe correct conformation relative to one another so that they retain thedesired activity. In one embodiment, the linker is from about 1 to 50amino acids in length, preferably about 1 to 30 amino acids in length.In one embodiment, linkers of 1 to 20 amino acids in length may be used,with from about 5 to about 10 amino acids finding use in someembodiments. Useful linkers include glycine-serine polymers, includingfor example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is aninteger of at least one (and generally from 3 to 4), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. While any suitable linker canbe used, many embodiments utilize a glycine-serine polymer, includingfor example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is aninteger of at least one (and generally from 3 to 4 to 5) as well as anypeptide sequence that allows for recombinant attachment of the twodomains with sufficient length and flexibility to allow each domain toretain its biological function. In some cases, and with attention beingpaid to “strandedness”, as outlined below, charged domain linkers, asused in some embodiments of scFv linkers can be used.

In some embodiments, the scFv linker is a charged scFv linker, a numberof which are shown in FIG. 14. Accordingly, the present inventionfurther provides charged scFv linkers, to facilitate the separation inpI between a first and a second monomer. That is, by incorporating acharged scFv linker, either positive or negative (or both, in the caseof scaffolds that use scFvs on different monomers), this allows themonomer comprising the charged linker to alter the pI without makingfurther changes in the Fc domains. These charged linkers can besubstituted into any scFv containing standard linkers. Again, as will beappreciated by those in the art, charged scFv linkers are used on thecorrect “strand” or monomer, according to the desired changes in pI. Forexample, as discussed herein, to make triple F format heterodimericantibody, the original pI of the Fv region for each of the desiredantigen binding domains are calculated, and one is chosen to make anscFv, and depending on the pI, either positive or negative linkers arechosen.

Charged domain linkers can also be used to increase the pI separation ofthe monomers of the invention as well, and thus those included in FIG.14 can be used in any embodiment herein where a linker is utilized.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein, particularly inthe Fc domains to allow either heterodimerization formation or thepurification of heterodimers away from homodimers. Full lengthantibodies generally include Fab and Fc domains, and can additionallycontain extra antigen binding domains such as scFvs, as is generallydepicted in the FIG. 1.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered to produceheterodimers, such as pI engineering. Other antibody fragments that canbe used include fragments that contain one or more of the CH1, CH2, CH3,hinge and CL domains of the invention that have been pI engineered. Forexample, Fc fusions are fusions of the Fc region (CH2 and CH3,optionally with the hinge region) fused to another protein. A number ofFc fusions are known the art and can be improved by the addition of theheterodimerization variants of the invention. In the present case,antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2; CH3;CH2 and CH3; CH1 and CH3, any or all of which can be made optionallywith the hinge region, utilizing any combination of heterodimerizationvariants described herein.

In particular, the formats depicted in FIG. 1 are antibodies, usuallyreferred to as “heterodimeric antibodies”, meaning that the protein hasat least two associated Fc sequences self-assembled into a heterodimericFc domain.

Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337;6,054,297; 6,407,213, all entirely incorporated by reference). Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654, entirely incorporated by reference. A variety of techniquesand methods for humanizing and reshaping non-human antibodies are wellknown in the art (See Tsurushita & Vasquez, 2004, Humanization ofMonoclonal Antibodies, Molecular Biology of B Cells, 533-545, ElsevierScience (USA), and references cited therein, all entirely incorporatedby reference). Humanization methods include but are not limited tomethods described in Jones et al., 1986, Nature 321:522-525; Riechmannet al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all entirelyincorporated by reference. Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated byreference.

In certain embodiments, the antibodies of the invention comprise a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally-occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a humanized antibodytypically is at least 90% identical in amino acids sequence to an aminoacid sequence encoded by a human germline immunoglobulin gene andcontains amino acid residues that identify the antibody as being derivedfrom human sequences when compared to the germline immunoglobulin aminoacid sequences of other species (e.g., murine germline sequences). Incertain cases, a humanized antibody may be at least 95, 96, 97, 98 or99%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a humanized antibody derived from aparticular human germline sequence will display no more than 10-20 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene (prior to the introduction of any skew, pIand ablation variants herein; that is, the number of variants isgenerally low, prior to the introduction of the variants of theinvention). In certain cases, the humanized antibody may display no morethan 5, or even no more than 4, 3, 2, or 1 amino acid difference fromthe amino acid sequence encoded by the germline immunoglobulin gene(again, prior to the introduction of any skew, pI and ablation variantsherein; that is, the number of variants is generally low, prior to theintroduction of the variants of the invention).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

IV. Heterodimeric Antibodies

Accordingly, in some embodiments the present invention providesheterodimeric antibodies that rely on the use of two different heavychain variant Fc domains that will self-assemble to form heterodimericantibodies.

The present invention is directed to novel constructs to provideheterodimeric antibodies that allow binding to more than one antigen orligand, e.g. to allow for bispecific binding. The heterodimeric antibodyconstructs are based on the self-assembling nature of the two Fc domainsof the heavy chains of antibodies, e.g. two “monomers” that assembleinto a “dimer”. Heterodimeric antibodies are made by altering the aminoacid sequence of each monomer as more fully discussed below. Thus, thepresent invention is generally directed to the creation of heterodimericantibodies which can co-engage antigens in several ways, relying onamino acid variants in the constant regions that are different on eachchain to promote heterodimeric formation and/or allow for ease ofpurification of heterodimers over the homodimers.

Thus, the present invention provides bispecific antibodies. An ongoingproblem in antibody technologies is the desire for “bispecific”antibodies that bind to two different antigens simultaneously, ingeneral thus allowing the different antigens to be brought intoproximity and resulting in new functionalities and new therapies. Ingeneral, these antibodies are made by including genes for each heavy andlight chain into the host cells. This generally results in the formationof the desired heterodimer (A-B), as well as the two homodimers (A-A andB-B (not including the light chain heterodimeric issues)). However, amajor obstacle in the formation of bispecific antibodies is thedifficulty in purifying the heterodimeric antibodies away from thehomodimeric antibodies and/or biasing the formation of the heterodimerover the formation of the homodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” or “skew” variants describedbelow and the “charge pairs” variants described below) as well as “pIvariants”, which allows purification of homodimers away fromheterodimers. As is generally described in WO2014/145806, herebyincorporated by reference in its entirety and specifically as below forthe discussion of “heterodimerization variants”, useful mechanisms forheterodimerization include “knobs and holes” (“KIH”; sometimes herein as“skew” variants (see discussion in WO2014/145806), “electrostaticsteering” or “charge pairs” as described in WO2014/145806, pI variantsas described in WO2014/145806, and general additional Fc variants asoutlined in WO2014/145806 and below.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric antibodies; one relies on theuse of pI variants, such that each monomer has a different pI, thusallowing the isoelectric purification of A-A, A-B and B-B dimericproteins. Alternatively, some scaffold formats, such as the “triple F”format, also allows separation on the basis of size. As is furtheroutlined below, it is also possible to “skew” the formation ofheterodimers over homodimers. Thus, a combination of stericheterodimerization variants and pI or charge pair variants findparticular use in the invention.

In general, embodiments of particular use in the present invention relyon sets of variants that include skew variants, that encourageheterodimerization formation over homodimerization formation, coupledwith pI variants, which increase the pI difference between the twomonomers.

Additionally, as more fully outlined below, depending on the format ofthe heterodimer antibody, pI variants can be either contained within theconstant and/or Fc domains of a monomer, or charged linkers, eitherdomain linkers or scFv linkers, can be used. That is, scaffolds thatutilize scFv(s) such as the Triple F format can include charged scFvlinkers (either positive or negative), that give a further pI boost forpurification purposes. As will be appreciated by those in the art, someTriple F formats are useful with just charged scFv linkers and noadditional pI adjustments, although the invention does provide pIvariants that are on one or both of the monomers, and/or charged domainlinkers as well. In addition, additional amino acid engineering foralternative functionalities may also confer pI changes, such as Fc, FcRnand KO variants.

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As is outlined more fully below, the pI changesof either or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine.). A number ofthese variants are shown in the Figures.

Accordingly, this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseit's pI (wt A−+B or wt A−−B), or by increasing one region and decreasingthe other region (A+−B− or A−B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIantibodies”) by incorporating amino acid substitutions (“pI variants” or“pI substitutions”) into one or both of the monomers. As shown herein,the separation of the heterodimers from the two homodimers can beaccomplished if the pIs of the two monomers differ by as little as 0.1pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in thepresent invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components, for example in thetriple F format, the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein.

Furthermore, as will be appreciated by those in the art and outlinedherein, in some embodiments, heterodimers can be separated fromhomodimers on the basis of size. As shown in FIG. 1 for example, severalof the formats allow separation of heterodimers and homodimers on thebasis of size.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying bispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and purification heterodimerization variants)are not included in the variable regions, such that each individualantibody must be engineered. In addition, in some embodiments, thepossibility of immunogenicity resulting from the pI variants issignificantly reduced by importing pI variants from different IgGisotypes such that pI is changed without introducing significantimmunogenicity. Thus, an additional problem to be solved is theelucidation of low pI constant domains with high human sequence content,e.g. the minimization or avoidance of non-human residues at anyparticular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric antibody production is important.

Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric antibodies in a variety of formats, which utilizeheterodimeric variants to allow for heterodimeric formation and/orpurification away from homodimers.

There are a number of suitable pairs of sets of heterodimerization skewvariants. These variants come in “pairs” of “sets”. That is, one set ofthe pair is incorporated into the first monomer and the other set of thepair is incorporated into the second monomer. It should be noted thatthese sets do not necessarily behave as “knobs in holes” variants, witha one-to-one correspondence between a residue on one monomer and aresidue on the other; that is, these pairs of sets form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25% homodimer A/A:50% heterodimer A/B:25%homodimer B/B).

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in FIG.10.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which areincorporated expressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

A list of suitable skew variants is found in FIG. 10, with FIG. 15showing some pairs of particular utility in many embodiments. Ofparticular use in many embodiments are the pairs of sets including, butnot limited to, Particularly preferred steric variant pairs include, butare not limited to, L368D/K370S:S364K, L368E/K370S:S364K,T411E/K360E/Q362E:D401K, L368D/K370S:S364K/E357L, K370S:S364K/E357Q andS364K/E357Q:L368D/K370S. In terms of nomenclature, the pair“S364K/E357Q:L368D/K370S” means that one of the monomers has the doublevariant set S364K/E357Q and the other has the double variant setL368D/K370S.

pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 11. As outlinedherein and shown in the figures, these changes are shown relative toIgG1, but all isotypes can be altered this way, as well as isotypehybrids. In the case where the heavy chain constant domain is fromIgG2-4, R133E and R133Q can also be used.

In one embodiment, for example in the bottle opener format, a preferredcombination of pI variants has one monomer (the negative Fab side)comprising 208D/295E/384D/418E/421D variants(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a secondmonomer (the positive scFv side) comprising a positively charged scFvlinker, including (GKPGS)₄. However, as will be appreciated by those inthe art, the first monomer includes a CH1 domain, including position208. Accordingly, in constructs that do not include a CH1 domain (forexample for heterodimeric Fc fusion proteins that do not utilize a CH1domain on one of the domains, for example in a dual scFv format), apreferred negative pI variant Fc set includes 295E/384D/418E/421Dvariants (Q295E/N384D/Q418E/N421D when relative to human IgG1).

Antibody Heterodimers Light Chain Variants

In the case of antibody based heterodimers, e.g. where at least one ofthe monomers comprises a light chain in addition to the heavy chaindomain, pI variants can also be made in the light chain. Amino acidsubstitutions for lowering the pI of the light chain include, but arenot limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E,K207E and adding peptide DEDE at the c-terminus of the light chain.Changes in this category based on the constant lambda light chaininclude one or more substitutions at R108Q Q124E, K126Q, N138D, K145Tand Q199E. In addition, increasing the pI of the light chains can alsobe done.

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIG. 21 of US Publ. 2014/0370013, hereby incorporated byreference. That is, IgG1 is a common isotype for therapeutic antibodiesfor a variety of reasons, including high effector function. However, theheavy constant region of IgG1 has a higher pI than that of IgG2 (8.10versus 7.31). By introducing IgG2 residues at particular positions intothe IgG1 backbone, the pI of the resulting monomer is lowered (orincreased) and additionally exhibits longer serum half-life. Forexample, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has aglutamic acid (pI 3.22); importing the glutamic acid will affect the pIof the resulting protein. As is described below, a number of amino acidsubstitutions are generally required to significant affect the pI of thevariant antibody. However, it should be noted as discussed below thateven changes in IgG2 molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 of US Pub.2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of the Fv and scaffold regions.Alternatively, the pI of each monomer can be compared.

pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, −7.4, induces the release of Fc backinto the blood. In mice, Dall'Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall'Acqua et al.2002, J. Immunol. 169:5171-5180, entirely incorporated by reference).The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid therelease of the Fc back into the blood. Therefore, the Fc mutations thatwill increase Fc's half-life in vivo will ideally increase FcRn bindingat the lower pH while still allowing release of Fc at higher pH. Theamino acid histidine changes its charge state in the pH range of 6.0 to7.4. Therefore, it is not surprising to find His residues at importantpositions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto

RIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. No.11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 2591, 428L/434S, 259I/308F, 436I/428L, 4361 or V/434S,436V/428L and 259I/308F/428L.

Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific antibodies that bindCD3 monovalently it is generally desirable to ablate FcγRIIIa binding toeliminate or significantly reduce ADCC activity. wherein one of the Fcdomains comprises one or more Fcγ receptor ablation variants. Theseablation variants are depicted in FIG. 12, and each can be independentlyand optionally included or excluded, with preferred aspects utilizingablation variants selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236del/5239K, E233P/L234V/L235A/G236del/5267K,E233P/L234V/L235A/G236del/5239K/A327G,E233P/L234V/L235A/G236del/5267K/A327G and E233P/L234V/L235A/G236del. Itshould be noted that the ablation variants referenced herein ablate FcγRbinding but generally not FcRn binding.

Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

Useful Formats of the Invention

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inFIG. 1. Some figures depict “single ended” configurations, where thereis one type of specificity on one “arm” of the molecule and a differentspecificity on the other “arm”. Other figures depict “dual ended”configurations, where there is at least one type of specificity at the“top” of the molecule and one or more different specificities at the“bottom” of the molecule. Thus, the present invention is directed tonovel immunoglobulin compositions that co-engage a different first and asecond antigen.

As will be appreciated by those in the art, the heterodimeric formats ofthe invention can have different valencies as well as be bispecific.That is, heterodimeric antibodies of the invention can be bivalent andbispecific, wherein CD3 is bound by one binding domain and PSMA is boundby a second binding domain. The heterodimeric antibodies can also betrivalent and bispecific, wherein the PSMA is bound by two bindingdomains and the CD3 by a second binding domain. As is outlined herein,it is preferable that the CD3 is bound only monovalently, to reducepotential side effects.

The present invention utilizes anti-CD3 antigen binding domains andanti-PSMA antigen binding domains. As will be appreciated by those inthe art, any collection of anti-CD3 CDRs, anti-CD3 variable light andvariable heavy domains, Fabs and scFvs as depicted in any of the Figures(see particularly FIGS. 2 through 7 and 21) can be used.

In some embodiments, the subject antibody includes an anti-CD3 antigenbinding domain that includes the variable light and variable heavydomains of anti-CD3 H1.30_L1.47 (FIG. 2). In some embodiments, thesubject antibody includes an anti-CD3 antigen binding domain thatincludes the vhCDR1-3 and vlCDR1-3 of anti-CD3 H1.30_L1.47 (FIG. 2). Insome embodiments, the subject antibody includes an anti-CD3_H1.30_L1.47scFv (FIG. 2).

In some embodiments, the subject antibody includes an anti-CD3 antigenbinding domain that includes the variable light and variable heavydomains of anti-CD3 H1.32_L1.47 (FIG. 3). In some embodiments, thesubject antibody includes an anti-CD3 antigen binding domain thatincludes the vhCDR1-3 and vlCDR1-3 of anti-CD3 H1.32_L1.47 (FIG. 3). Insome embodiments, the subject antibody includes an anti-CD3 H1.32_L1.47scFv (FIG. 3).

In some embodiments, the subject antibody includes an anti-CD3 antigenbinding domain that includes the variable light and variable heavydomains of anti-CD3 H1.89_L1.47 (FIG. 4). In some embodiments, thesubject antibody includes an anti-CD3 antigen binding domain thatincludes the vhCDR1-3 and vhCDR1-3 of anti-CD3 H1.89_L1.47 (FIG. 4). Insome embodiments, the subject antibody includes an anti-CD3 H1.89_L1.47scFv (FIG. 4).

In some embodiments, the subject antibody includes an anti-CD3 antigenbinding domain that includes the variable light and variable heavydomains of anti-CD3 H1.90_L1.47 (FIG. 5). In some embodiments, thesubject antibody includes an anti-CD3 antigen binding domain thatincludes the vhCDR1-3 and vhCDR1-3 of anti-CD3 H1.90_L1.47 (FIG. 5). Insome embodiments, the subject antibody includes an anti-CD3 H1.90_L1.47scFv (FIG. 5).

In some embodiments, the subject antibody includes an anti-CD3 antigenbinding domain that includes the variable light and variable heavydomain of anti-CD3 H1.33_L1.47 (FIG. 6). In some embodiments, thesubject antibody includes an anti-CD3 antigen binding domain thatincludes the vhCDR1-3 and vlCDR1-3 of anti-CD3 H1.33_L1.47 (FIG. 6). Insome embodiments, the subject antibody includes an anti-CD3 H1.33_L1.47scFv (FIG. 6).

In some embodiments, the subject antibody includes an anti-CD3 antigenbinding domain that includes the variable light and variable heavydomain of anti-CD3 H1.31_L1.47 (FIG. 7). In some embodiments, thesubject antibody includes an anti-CD3 antigen binding domain thatincludes the vhCDR1-3 and vlCDR1-3 of anti-CD3 H1.31_L1.47 (FIG. 7). Insome embodiments, the subject antibody includes an anti-CD3 H1.31_L1.47scFv (FIG. 7).

Similarly, any of the anti-PSMA antigen binding domains, whetheranti-PSMA CDRs, anti-PSMA variable light and variable heavy domains,Fabs and scFvs as depicted in FIGS. 8 and 20 can be used, optionally andindependently combined in any combination. In some embodiments, thesubject antibody includes an anti-PSMA antigen binding domain thatincludes the variable light and variable heavy domains of anti-PSMA J591H1_L1 (FIG. 20). In some embodiments, the subject antibody includes ananti-PSMA antigen binding domain that includes the vhCDR1-3 and vhCDR1-3of anti-PSMA J591 H1_L1 (FIG. 20). In some embodiments, the subjectantibody includes an anti-PSMA J591 H1_L1 scFv having the variable lightand variable heavy domains or vhCDR1-3 and vhCDR1-3 of anti-PSMA J591H1_L1 (FIG. 20).

Bottle Opener Format

One heterodimeric scaffold that finds particular use in the presentinvention is the “triple F” or “bottle opener” scaffold format as shownin FIG. 1A. In this embodiment, one heavy chain of the antibody containsan single chain Fv (“scFv”, as defined below) and the other heavy chainis a “regular” FAb format, comprising a variable heavy chain and a lightchain. This structure is sometimes referred to herein as “triple F”format (scFv-FAb-Fc) or the “bottle-opener” format, due to a roughvisual similarity to a bottle-opener (see FIG. 1). The two chains arebrought together by the use of amino acid variants in the constantregions (e.g. the Fc domain, the CH1 domain and/or the hinge region)that promote the formation of heterodimeric antibodies as is describedmore fully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.).

Many of the embodiments outlined herein rely in general on the bottleopener format that comprises a first monomer comprising an scFv,comprising a variable heavy and a variable light domain, covalentlyattached using an scFv linker (charged, in many instances), where thescFv is covalently attached to the N-terminus of a first Fc domainusually through a domain linker (which, as outlined herein can either beun-charged or charged). The second monomer of the bottle opener formatis a heavy chain, and the composition further comprises a light chain.

In general, in many preferred embodiments, the scFv is the domain thatbinds to the CD3, with the Fab of the heavy and light chains binding toPSMA. In addition, the Fc domains of the invention generally compriseskew variants (e.g. a set of amino acid substitutions as shown in FIGS.10 and 15, with particularly useful skew variants being selected fromthe group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L andK370S:S 364K/E357Q), optionally ablation variants, and the heavy chaincomprises pI variants.

In some embodiments, any of the vh and vl sequences depicted herein(including all anti-PSMA and anti-CD3 vh and vl sequences and relatedsCDR sequences depicted in the Figure) can be added to the bottle openerbackbone formats of FIG. 25 as the “Fab side”, using any of the anti-CD3scFv sequences shown in the Figures. Anti-CD3 sequences findingparticular use in these embodiments are anti-CD3 H1.30_L1.47, anti-CD3H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3H1.33_L1.47 and anti-CD3 H1.31_L1.47 (vh, vl and CDR sequences depictedin FIGS. 2,-7, respectively), attached as the scFv side of the backbonesshown in FIG. 25.

The present invention provides bottle opener formats where the anti-CD3scFv sequences are as shown in FIGS. 2 to 7 and 21, including anycombination with the backbone formats of FIG. 25. In addition, any ofthe anti-CD3 vh, vl and CDR sequences as shown in FIGS. 2 to 7 and 21can be used as the Fab side.

The present invention provides bottle opener formats wherein theanti-PSMA sequences are as shown in FIGS. 8 and 20. Similar to theabove, each anti-PSMA vh, vl, and related CDRs sequences can be eitherthe Fab side or the scFv side, and can be linked as one of the antigenbinding domains of a bottle opener format, including those of FIG. 25.When the anti-PSMA sequences are the Fab side, any anti-CD3 scFvsequences and related vh, vl and CDR sequences set forth in the Figurescan be used, particularly including anti-CD3 H1.30_L1.47, anti-CD3H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3H1.33_L1.47 and anti-CD3 H1.31_L1.47, attached as the scFv side of thebackbones shown in FIG. 25.

In some embodiments, the subject antibody is a bottle opener formatantibody having a Fab-Fc heavy chain scaffold, a scFv-Fc heavy chainscaffold and light chain scaffold as shown in FIG. 21E. Any variableheavy domain, including, but not limited to, any of the variable heavydomains described herein can be operably linked to the Fab-Fc heavychain scaffold described in FIG. 21E. Any variable light domain,including, but not limited to, any of the variable light domainsdescribed herein can be operably linked to the light chain scaffolddescribed in FIG. 21E.

In some embodiments, the variable heavy domain and the variable lightdomain operably linked to the Fab-Fc heavy chain scaffold and lightchain scaffold (FIG. 21E), respectively, are variable heavy and lightdomains of an anti-PSMA binding domain. In some embodiments, thevariable heavy domain and the variable light domain includes thevariable light and variable heavy domains of anti-PSMA J591 H1_L1 (FIG.20). In some embodiments, the variable heavy domain includes thevhCDR1-3 of J591 H1_L1 (FIG. 20) and the variable light domain includesthe vhCDR1-3 of J591 H1_L1 (FIG. 20).

In some embodiments, the variable heavy domain and the variable lightdomain operably linked to the Fab-Fc heavy chain scaffold and lightchain scaffold (FIG. 21E), respectively, are variable heavy and lightdomains of an anti-CD3 binding domain. In some embodiments, the variableheavy domain and the variable light domain includes the variable lightand variable heavy domains of anti-CD3 H1.30_L1.47 (FIG. 2), anti-CD3H1.32_L1.47 (FIG. 3), anti-CD3 H1.89_L1.47 (FIG. 4), H1.90_L1.47 (FIG.5), H1.33_L1.47 (FIG. 6), or H1.31_L1.47 (FIG. 7). In some embodiments,the variable heavy domain includes the vhCDR1-3 of anti-CD3 H1.30_L1.47(FIG. 2), anti-CD3 H1.32_L1.47 (FIG. 3), anti-CD3 H1.89_L1.47 (FIG. 4),H1.90_L1.47 (FIG. 5), H1.33_L1.47 (FIG. 6), or H1.31_L1.47 (FIG. 7) andthe variable light domain includes the vlCDR1-3 of anti-CD3 H1.30_L1.47(FIG. 2), anti-CD3 H1.32_L1.47 (FIG. 3), anti-CD3 H1.89_L1.47 (FIG. 4),H1.90_L1.47 (FIG. 5), H1.33_L1.47 (FIG. 6), or H1.31_L1.47 (FIG. 7),respectively (e.g., the variable heavy domain includes the vhCDR1-3 ofanti-CD3 H1.30_L1.47 and the variable light domain includes the vlCDR1-3of anti-CD3 H1.30_L1.47).

In some embodiments, the scFv-Fc heavy chain scaffold (FIG. 21E) isoperably linked to an anti-CD3 scFv. In some embodiments, the anti-CD3scFv includes the variable heavy and variable light domains ofH1.30_L1.47 (FIG. 2), H1.32_L1.47 (FIG. 3), H1.89_L1.47 (FIG. 4),H1.90_L1.47 (FIG. 5), H1.33_L1.47 (FIG. 6) or H1.31_L1.47 (FIG. 7). Insome embodiments, the anti-CD3 scFv includes the vhCDR1-3 and vlCDR1-3of H1.30_L1.47 (FIG. 2), H1.32_L1.47 (FIG. 3), H1.89_L1.47 (FIG. 4),H1.90_L1.47 (FIG. 5), H1.33_L1.47 (FIG. 6) or H1.31_L1.47 (FIG. 7). Insome embodiments, the anti-CD3 scFv is an H1.30_L1.47 scFv (FIG. 2),H1.32_L1.47 scFv (FIG. 3), H1.89_L1.47 scFv (FIG. 4), H1.90_L1.47 scFv(FIG. 5), H1.33_L1.47 scFv (FIG. 6) or H1.31_L1.47 scFv (FIG. 7)

In some embodiments, the scFv-Fc heavy chain scaffold (FIG. 21E) isoperably linked to an anti-PSMA scFv. In some embodiments, the anti-PSMAscFv includes the variable heavy domain and variable light domain ofanti-PSMA J591 H1_L1 (FIG. 20). In some embodiments, the scFv includesthe vhCDR1-3 of J591 H1_L1 (FIG. 20) and the vlCDR1-3 of J591 H1_L1(FIG. 20). In some embodiments the anti-PSMA scFv is an J591 H1_L1 scFv(FIG. 20).

In certain embodiments, the subject anti-PSMA x anti x CD3 antibody hasthe sequence of any one of the bottle opener formats depicted in FIGS.9, 21A-D and 25.

mAb-Fv Format

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-Fv format shown in FIG. 1. In this embodiment, theformat relies on the use of a C-terminal attachment of an “extra”variable heavy domain to one monomer and the C-terminal attachment of an“extra” variable light domain to the other monomer, thus forming a thirdantigen binding domain, wherein the Fab portions of the two monomersbind PSMA and the “extra” Fv domain binds CD3.

In this embodiment, the first monomer comprises a first heavy chain,comprising a first variable heavy domain and a first constant heavydomain comprising a first Fc domain, with a first variable light domaincovalently attached to the C-terminus of the first Fc domain using adomain linker. The second monomer comprises a second variable heavydomain, a second constant heavy domain comprising a second Fc domain,and a third variable heavy domain covalently attached to the C-terminusof the second Fc domain using a domain linker. In some embodiments, thetwo C-terminally attached variable domains make up a Fv that binds CD3(as it is less preferred to have bivalent CD3 binding). This embodimentfurther utilizes a common light chain comprising a variable light domainand a constant light domain that associates with the heavy chains toform two identical Fabs that bind PSMA.

In some embodiments, the first heavy chain includesvh1-CH1-hinge-CH2-CH3-[optional linker]-vl2 and the second monomerincludes vh1-CH1-hinge-CH2-CH3-[optional linker]-vh2 and the commonlight chain includes vl1. In this embodiment, vh1 and vl1 form PSMAbinding domains and vh2 and vl2 form a CD3 binding domain.

As for many of the embodiments herein, these constructs include skewvariants, pI variants, ablation variants, additional Fc variants, etc.as desired and described herein.

The present invention provides mAb-Fv formats where the anti-CD3 scFvsequences are shown in FIG. 2 to FIG. 7.

The present invention provides bottle opener formats wherein theanti-PSMA sequences are as shown in FIG. 8 and FIG. 20.

The present invention provides mAb-Fv formats comprising ablationvariants as shown in FIG. 12.

The present invention provides mAb-Fv formats comprising skew variantsas shown in FIG. 10 and FIG. 15.

mAb-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-scFv format shown in FIG. 1. In this embodiment,the format relies on the use of a C-terminal attachment of a scFv to oneof the monomers, thus forming a third antigen binding domain, whereinthe Fab portions of the two monomers bind PSMA and the “extra” scFvdomain binds CD3. Thus, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aC-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation (vh1-CH1-hinge-CH2-CH3-[optional linker]-vh2-scFv linker-vl2or vh1-CH1-hinge-CH2-CH3-[optional linker]-vl2-scFv linker-vh2). Thisembodiment further utilizes a common light chain comprising a variablelight domain and a constant light domain that associates with the heavychains to form two identical Fabs that bind PSMA. As for many of theembodiments herein, these constructs include skew variants, pI variants,ablation variants, additional Fc variants, etc. as desired and describedherein.

The present invention provides mAb-scFv formats where the anti-CD3 scFvsequences are as shown in FIG. 2 to FIG. 7.

The present invention provides mAb-scFv formats wherein the anti-PSMAsequences are as shown in in FIG. 8 and FIG. 20.

The present invention provides mAb-scFv formats comprising ablationvariants as shown in FIG. 12.

The present invention provides mAb-scFv formats comprising skew variantsas shown in FIG. 10 and FIG. 15.

Central scFv Format

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-scFv format shown in FIG. 1. In thisembodiment, the format relies on the use of an inserted scFv domain thusforming a third antigen binding domain, wherein the Fab portions of thetwo monomers bind PSMA and the “extra” scFv domain binds CD3. The scFvdomain is inserted between the Fc domain and the CH1-Fv region of one ofthe monomers, thus providing a third antigen binding domain.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain and Fc domain (and optionalhinge), with a scFv comprising a scFv variable light domain, an scFvlinker and a scFv variable heavy domain. The scFv is covalently attachedbetween the C-terminus of the CH1 domain of the heavy constant domainand the N-terminus of the first Fc domain using optional domain linkers(e.g., vh1-CH1-[optional linker]-vh2-scFv linker-vl2-[optional linkerincluding the hinge]-CH2-CH3 or vh1-CH1-[optional linker]-vl2-scFvlinker-vh2-[optional linker including the hinge]-CH2-CH3. The secondmonomer is a standard Fab side (e.g., vh1-CH1-[optional linker includingthe hinge]-CH2-CH3). This embodiment further utilizes a common lightchain comprising a variable light domain (e.g., vl1) and a constantlight domain, that associates with the heavy chains to form twoidentical Fabs that bind PSMA. As for many of the embodiments herein,these constructs include skew variants, pI variants, ablation variants,additional Fc variants, etc. as desired and described herein.

The present invention provides Central-scFv formats where the anti-CD3scFv sequences are as shown in FIGS. 2 to 7.

The present invention provides Central-scFv formats wherein theanti-PSMA sequences are as shown in FIG. 8 and FIG. 20

The present invention provides Central-scFv formats comprising ablationvariants as shown in FIG. 12.

The present invention provides Central-scFv formats comprising skewvariants as shown in FIG. 10 and FIG. 15.

Central-Fv Format

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-Fv format shown in FIG. 1. In this embodiment,the format relies on the use of an inserted scFv domain thus forming athird antigen binding domain, wherein the Fab portions of the twomonomers bind PSMA and the “extra” Fv domain binds CD3. The scFv domainis inserted between the Fc domain and the CH1-Fv region of the monomers,thus providing a third antigen binding domain, wherein each monomercontains a component of the Fv (e.g., one monomer comprises a variableheavy domain and the other a variable light domain).

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain and Fc domain and anadditional variable light domain. The additional variable light domainis covalently attached between the C-terminus of the CH1 domain of theheavy constant domain and the N-terminus of the first Fc domain usingdomain linkers (e.g., vh1-CH1-[optional linker]-vl2-hinge-CH2-CH3). Theother monomer comprises a first heavy chain comprising a first variableheavy domain, a CH1 domain and Fc domain and an additional variableheavy domain (e.g., vh1-CH1-[optional linker]-vh2-hinge-CH2-CH3). Theadditional variable heavy domain is covalently attached between theC-terminus of the CH1 domain of the heavy constant domain and theN-terminus of the first Fc domain using domain linkers.

This embodiment further utilizes a common light chain comprising avariable light domain and a constant light domain, that associates withthe heavy chains to form two identical Fabs that bind PSMA. As for manyof the embodiments herein, these constructs include skew variants, pIvariants, ablation variants, additional Fc variants, etc. as desired anddescribed herein.

The present invention provides Central-scFv formats where the anti-CD3scFv sequences are as shown in FIGS. 2 to 7.

The present invention provides Central-scFv formats wherein theanti-PSMA sequences are as shown in FIG. 8 and FIG. 20.

The present invention provides Central-scFv formats comprising ablationvariants as shown in FIG. 12.

The present invention provides Central-scFv formats comprising skewvariants as shown in FIG. 10 and FIG. 15.

One Armed Central-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the one armed central-scFv format shown in FIG. 1. In thisembodiment, one monomer comprises just an Fc domain, while the othermonomer uses an inserted scFv domain thus forming the second antigenbinding domain. In this format, either the Fab portion binds PSMA andthe scFv binds CD3 or vice versa. The scFv domain is inserted betweenthe Fc domain and the CH1-Fv region of one of the monomers.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain and Fc domain, with a scFvcomprising a scFv variable light domain, an scFv linker and a scFvvariable heavy domain. The scFv is covalently attached between theC-terminus of the CH1 domain of the heavy constant domain and theN-terminus of the first Fc domain using domain linkers (e.g.,vh1-CH1-[optional linker]-vh2-scFv linker-vl2-[optional linker includingthe hinge]-CH2-CH3 or vh1-CH1-[optional linker]-vl2-scFvlinker-vh2-[optional linker including the hinge]-CH2-CH3). The secondmonomer comprises an Fc domain (CH2-CH3). This embodiment furtherutilizes a light chain comprising a variable light domain and a constantlight domain, that associates with the heavy chain to form a Fab. As formany of the embodiments herein, these constructs include skew variants,pI variants, ablation variants, additional Fc variants, etc. as desiredand described herein.

The present invention provides one armed Central-scFv formats where theanti-CD3 scFv sequences are as shown in FIG. 2 to FIG. 7.

The present invention provides one armed Central-scFv formats whereinthe anti-PSMA sequences are as shown in FIG. 8 and FIG. 20.

The present invention provides one armed Central-scFv formats comprisingablation variants as shown in FIG. 12.

The present invention provides one armed Central-scFv formats comprisingskew variants as shown in FIG. 10 and FIG. 15.

Dual scFv Formats

The present invention also provides dual scFv formats as are known inthe art and shown in FIG. 1. In particular, the invention provides dualscFv formats where the anti-CD3 scFv sequences are as shown in FIG. 2 toFIG. 7.

The present invention provides dual scFv formats wherein the anti-PSMAsequences are as shown in FIG. 8. In some of the embodiments, theanti-PSMA and anti-CD3 scFv includes an scFv linker. In certainembodiments, the scFv linker is an scFv linker shown in FIG. 14.

The present invention provides dual scFv formats comprising ablationvariants as shown in FIG. 12.

The present invention provides dual scFv formats comprising skewvariants as shown in FIG. 10 and FIG. 15.

Common Light Chain Formats

One heterodimeric scaffold that finds particular use in the presentinvention is the common light chain format shown in FIG. 1. In thisembodiment, the first monomer comprises a first heavy chain, comprisinga first variable heavy domain and a first constant heavy domaincomprising a first Fc domain. The second monomer comprises a secondvariable heavy domain and a second constant heavy domain comprising asecond Fc domain. Each of the first and second monomers are associatedwith a common light chain comprising a variable light domain and aconstant light domain. As for many of the embodiments herein, theseconstructs include skew variants, pI variants, ablation variants,additional Fc variants, etc. as desired and described herein.

The present invention provides common light chain formats where theanti-CD3 sequences are as shown in FIG. 2 to FIG. 7.

The present invention provides common light chain formats wherein theanti-PSMA sequences are as shown in FIG. 8 and FIG. 20.

The present invention provides common light chain formats comprisingablation variants as shown in FIG. 12.

The present invention provides common light chain formats comprisingskew variants as shown in FIG. 10 and FIG. 15.

Bispecific IgG

One heterodimeric scaffold that finds particular use in the presentinvention is the bispecific format shown in FIG. 1. In this embodiment,the first monomer comprises a first heavy chain, comprising a firstvariable heavy domain and a first constant heavy domain comprising afirst Fc domain. The second monomer comprises a second variable heavydomain and a second constant heavy domain comprising a second Fc domain.Each of the first and second monomers are associated with a light chain(i.e., a first light chain and a second light chain, respectively)comprising a variable light domain and a constant light domain. As formany of the embodiments herein, these constructs include steric variants(e.g., knobs into holes variants), skew variants, pI variants, ablationvariants, additional Fc variants, etc. as desired and described herein.

The present invention provides bispecific IgG formats where the anti-CD3sequences are as shown in FIG. 2 to FIG. 7.

The present invention provides bispecific IgG formats wherein theanti-PSMA sequences are as shown in FIG. 8 and FIG. 20.

The present invention provides bispecific IgG formats comprisingablation variants as shown in FIG. 12.

The present invention provides bispecific IgG formats comprising skewvariants as shown in FIG. 10 and FIG. 15.

Nucleic Acids of the Invention

The invention further provides nucleic acid compositions encoding thebispecific antibodies of the invention. As will be appreciated by thosein the art, the nucleic acid compositions will depend on the format andscaffold of the heterodimeric protein. Thus, for example, when theformat requires three amino acid sequences, such as for the triple Fformat (e.g. a first amino acid monomer comprising an Fc domain and ascFv, a second amino acid monomer comprising a heavy chain and a lightchain), three nucleic acid sequences can be incorporated into one ormore expression vectors for expression. Similarly, some formats (e.g.dual scFv formats such as disclosed in FIG. 1) only two nucleic acidsare needed; again, they can be put into one or two expression vectors.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the heterodimericantibodies of the invention. Generally the nucleic acids are operablylinked to any number of regulatory elements (promoters, origin ofreplication, selectable markers, ribosomal binding sites, inducers,etc.). The expression vectors can be extra-chromosomal or integratingvectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g. CHO cells), finding use in manyembodiments.

In some embodiments, nucleic acids encoding each monomer and theoptional nucleic acid encoding a light chain, as applicable depending onthe format, are each contained within a single expression vector,generally under different or the same promoter controls. In embodimentsof particular use in the present invention, each of these two or threenucleic acids are contained on a different expression vector. As shownherein and in 62/025,931, hereby incorporated by reference, differentvector ratios can be used to drive heterodimer formation. That is,surprisingly, while the proteins comprise first monomer:secondmonomer:light chains (in the case of many of the embodiments herein thathave three polypeptides comprising the heterodimeric antibody) in a1:1:2 ratio, these are not the ratios that give the best results.

The heterodimeric antibodies of the invention are made by culturing hostcells comprising the expression vector(s) as is well known in the art.Once produced, traditional antibody purification steps are done,including an ion exchange chromotography step. As discussed herein,having the pIs of the two monomers differ by at least 0.5 can allowseparation by ion exchange chromatography or isoelectric focusing, orother methods sensitive to isoelectric point. That is, the inclusion ofpI substitutions that alter the isoelectric point (pI) of each monomerso that such that each monomer has a different pI and the heterodimeralso has a distinct pI, thus facilitating isoelectric purification ofthe “triple F” heterodimer (e.g., anionic exchange columns, cationicexchange columns). These substitutions also aid in the determination andmonitoring of any contaminating dual scFv-Fc and mAb homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

Treatments

Once made, the compositions of the invention find use in a number ofapplications, including the treatment of cancers that express oroverexpress PSMA, including, but not limited to, prostate cancer.

Accordingly, the heterodimeric compositions of the invention find use inthe treatment of these cancers.

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecifcities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the bispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an bispecific antibody used in the present invention is about 0.1-100mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, suchas about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about1, or about 3 mg/kg. In another embodiment, the antibody is administeredin a dose of 1 mg/kg or more, such as a dose of from 1 to 20 mg/kg, e.g.a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the bispecific antibody is administered by infusionin a weekly dosage of from 10 to 500 mg/kg such as of from 200 to 400mg/kg Such administration may be repeated, e.g., 1 to 8 times, such as 3to 5 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the bispecific antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the bispecific antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the bispecific antibody.

In a further embodiment, the bispecific antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the bispecific antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the bispecific antibody is administered by a regimenincluding one infusion of an bispecific antibody followed by an infusionof an bispecific antibody conjugated to a radioisotope. The regimen maybe repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the bispecific antibody molecule thereof is used incombination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein.

Example 1: Bispecifics Production

Amino acid sequences for anti-PSMA x anti-CD3 bispecifics are listed inFIGS. 9, and 21A-D. DNA encoding the three chains needed for bispecificexpression were generated by gene synthesis (Blue Heron Biotechnology,Bothell, Wash.) and were subcloned using standard molecular biologytechniques into the expression vector pTT5. Substitutions wereintroduced using either site-directed mutagenesis (QuikChange,Stratagene, Cedar Creek, Tex.) or additional gene synthesis andsubcloning. DNA was transfected into HEK293E cells for expression andresulting proteins were purified from the supernatant using protein Aaffinity (GE Healthcare) and cation exchange chromatography. Cationexchange chromatography purification was performed using a HiTrap SP HPcolumn (GE Healthcare) with a wash/equilibration buffer of 50 mM MES, pH6.0 and an elution buffer of 50 mM MES, pH 6.0+1 M NaCl linear gradient.

The PSMA affinities (KD) for exemplary antibodies XENP14484 andXENP19722 were measured by biolayer interferometry (BLI) on an Octet Redinstrument (ForteBio)(FIG. 22). Bispecifics (XENP14484, XENP19722) werecaptured onto an AHC sensor, followed by human or cynomolgus monkey PSMAat concentrations of 50 to 0.78125 nM (two-fold dilutions).

Example 2: Redirected T Cell Cytotoxicity

Anti-PSMA x anti-CD3 bispecifics were characterized in vitro for cellsurface binding to and redirected T cell cytotoxicity (RTCC) of thePSMA′ LNCaP prostate cancer cell line (FIGS. 16 and 23). Assay detailsare indicated in the figure legend. As shown in these figures, subjectanti-PSMA x anti-CD3 antibodies XENP14484, and XENP19722 were capable ofbinding to LNCaP prostate cancer cells and mediating RTCC killing invitro, whereas anti-RSV control XENP13245 did not PSMA exhibit or RTCC.

Example 3: Cynomolgus Monkey Pharmacology

Cynomolgus monkeys (n=3) were given a single dose of either XENP14484(anti-PSMA x anti-CD3, 30 μg/kg) or XENP13245 (anti-RSV x CD3, 3 mg/kg).CD4′ T cell margination and activation by CD69 MFI were examined by flowcytometry. IL-6 serum levels were examined by ELISA (FIG. 17A). Inaddition, CD8′ T cell margination and activation by CD69 MFI wereexamined by flow cytometry and TNFα serum levels were examined by ELISA(FIG. 17B). As shown in these figures, T cell response and CytokineRelease Syndrome (CRS) is dependent on the engagement of target cells bythe anti-PSMA x anti-CD3 antibody in monkeys.

Example 4: CD3 Affinity and Cytokine Release Syndrome

Cynomolgus monkey Cynomolgus monkeys (n=3) were administered a singleintraveneous (i.v.) dose of either 0.06 mg/kg XENP14484 (high CD3affinity) or 1 mg/kg XENP19722 (low CD3 affinity). As shown in FIG. 24,XENP14484 and XENP19722 antibodies exhibit similar T cell marginationand activation (CD4+(top panel) and CD8+(middle panel)) despite havingdifferent CD3 affinities. Despite similarities in T cell activation,however, XENP19722, which has a low affinity to CD3, induced lesscytokine reduced syndrome (CRS) compared to XENP14484, which has a highaffinity to CD3 (FIG. 24, bottom panel). Such lower CD3 affinitycorrelates with reduced cytokine release even at higher dose levels. Assuch, the potency of anti-PSMA bispecifics can be modulated by tuningtheir ability to recruit T cells.

1.-56. (canceled)
 57. A composition comprising a prostate specificmembrane antigen (PSMA) binding domain, wherein said anti-PSMA bindingdomain comprises: a) a variable heavy domain comprising a vhCDR1 havingSEQ ID NO: 111, a vhCDR2 having SEQ ID NO: 112, and a vhCDR3 having SEQID NO: 113; and b) a variable light domain comprising a vhCDR1 havingSEQ ID NO: 107, a vhCDR2 having SEQ ID NO: 108, and a vhCDR3 having SEQID NO:
 109. 58. A composition according to claim 57, wherein saidvariable heavy variable domain comprises amino acid sequence SEQ ID NO:110 and said variable light domain comprises amino acid sequence SEQ IDNO:
 106. 59. A nucleic acid composition comprising: a) a first nucleicacid encoding said variable heavy domain of claim 57; and b) a secondnucleic acid encoding said variable light domain of claim
 57. 60. Anexpression vector composition comprising: a) a first expression vectorcomprising said first nucleic acid of claim 59; and b) a secondexpression vector comprising said second nucleic acid of claim
 59. 61. Ahost cell comprising said expression vector composition of claim
 60. 62.A heterodimeric antibody comprising: a) a first monomer comprising ascFv-linker-CH2-CH3, wherein CH2-CH3 is a first Fc domain, and whereinscFv is a single chain FIT region that binds a first antigen b) a secondmonomer comprising a VH-CH1-hinge-CH2-CH3, wherein VH is a variableheavy domain and CH2-CH3 is a second Fc domain; and c) a light chaincomprising a variable light domain and a constant light domain; whereinsaid variable heavy domain comprises a vhCDR1 having SEQ ID NO: 111, avhCDR2 having SEQ ID NO: 112, and a vhCDR3 having SEQ ID NO: 113, saidvariable light domain comprises a vhCDR1 having SEQ ID NO: 107, a vhCDR2having SEQ ID NO: 108, and a vhCDR3 having SEQ ID NO: 109, and whereinsaid variable heavy domain and said variable light domain form a PSMAbinding domain.
 63. A heterodimeric antibody according to claim 62,where said variable heavy domain comprises amino acid sequence SEQ IDNO: 110 and said variable light domain comprises amino acid sequence SEQID NO:
 106. 64. A heterodimeric antibody according to claim 62, whereinsaid first Fc domain and said second Fc domain comprise a set of aminoacid substitutions selected from the group consisting ofS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L and K370S:S364K/E357Q.65. A heterodimeric antibody according to claim 62, wherein said scFvcomprises a charged linker.
 66. A heterodimeric antibody according toclaim 62, wherein said CH1-hinge-CH2-CH3 of said second monomercomprises the amino acid substitutions N208D/Q295E/N384D/Q418E/N421D.67. A heterodimeric antibody according to claim 62, wherein said firstand second Fc domains each comprise the amino acid substitutionsE233P/L234V/L235A/G236del/S267K.
 68. A heterodimeric antibody accordingto claim 62, wherein said first Fc domain comprises amino acid variantsS364K/E357Q, wherein said second Fc domain comprises amino acid variantsL368D/K370S, wherein said first and second Fc domains each comprisesamino acid variants E233P/L234V/L235A/G236del/S267K, and wherein saidCH1-hinge-CH2-CH3 of the second monomer comprises amino acid variantsN208D/Q295E/N384D/Q418E/N421D.
 69. A nucleic acid compositioncomprising: a) a first nucleic acid encoding said first monomer of claim62; b) a second nucleic acid encoding said second monomer of claim 62;and c) a third nucleic acid encoding said light chain of claim
 62. 70.An expression vector composition comprising: a) a first expressionvector comprising said first nucleic acid of claim 69; b) a secondexpression vector comprising said second nucleic acid of claim 69; andc) a third expression vector comprising said third nucleic acid of claim69.
 71. A host cell comprising said nucleic acid composition of claim69.
 72. A host cell comprising said expression vector composition ofclaim
 70. 73. A method of making a heterodimeric antibody comprisingculturing said host cell of claim 71 or 72 under conditions wherein saidantibody is expressed, and recovering said heterodimeric antibody.
 74. Aheterodimeric antibody comprising: a) a first monomer comprising aVH1-CH1-linker 1-scFv-linker 2-CH2-CH3, wherein VH is a variable heavydomain, linker 1 and linker 2 are a first domain linker and seconddomain linker, respectively, and CH2-CH3 is a first Fc domain; b) asecond monomer comprising a VH-CH1-hinge-CH2-CH3, wherein VH is avariable heavy domain and CH2-CH3 is a second Fc domain; and c) a commonlight chain comprising a variable light domain and a constant lightdomain; wherein said variable heavy domain of said first monomer andsaid variable light domain form a first PSMA binding domain, and saidvariable heavy domain of said second monomer and said variable lightdomain form a second PSMA binding domain, wherein said variable heavydomain of said first monomer and said variable heavy domain of saidsecond monomer each comprise a vhCDR1 having SEQ ID NO: 111, a vhCDR2having SEQ ID NO: 112, and a vhCDR3 having SEQ ID NO: 113, and saidvariable light domain comprises a vhCDR1 having SEQ ID NO: 107, a vhCDR2having SEQ ID NO: 108, and a vhCDR3 having SEQ ID NO:
 109. 75. Aheterodimeric antibody according to claim 74, where said variable heavydomain of said first monomer and said variable heavy domain of saidsecond monomer each comprise amino acid sequence SEQ ID NO: 110 and saidvariable light domain comprises amino acid sequence SEQ ID NO:
 106. 76.A heterodimeric antibody according to claim 74, wherein said first Fcdomain and said second Fc domain comprise a set of variants selectedfrom the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L andK370S:S364K/E357Q.
 77. A heterodimeric antibody according to claim 74,wherein said CH1-hinge-CH2-CH3 of said second monomer comprises theamino acid substitutions N208D/Q295E/N384D/Q418E/N421D.
 78. Aheterodimeric antibody according to claim 74, wherein said first andsecond Fc domains each comprise the amino acid substitutionsE233P/L234V/L235A/G236del/S267K.
 79. A heterodimeric antibody accordingto claim 74, wherein said first Fc domain comprises amino acid variantsS364K/E357Q, wherein said second Fc domain comprises amino acid variantsL368D/K370S, wherein said first and second Fc domains each comprisesamino acid variants E233P/L234V/L235A/G236del/S267K, and wherein saidCH1-hinge-CH2-CH3 of the second monomer comprises amino acid variantsN208D/Q295E/N384D/Q418E/N421D.
 80. A nucleic acid compositioncomprising: a) a first nucleic acid encoding said first monomer of claim74; b) a second nucleic acid encoding said second monomer of claim 74;and c) a third nucleic acid encoding said light chain of claim
 74. 81.An expression vector composition comprising: a) a first expressionvector comprising said first nucleic acid of claim 80; b) a secondexpression vector comprising said second nucleic acid of claim 80; andc) a third expression vector comprising said third nucleic acid of claim80.
 82. A host cell comprising the nucleic acid composition of claim 80.83. A host cell comprising the expression vector composition of claim81.
 84. A method of making a heterodimeric antibody comprising culturingthe host cell of claim 82 or 83 under conditions wherein said antibodyis expressed, and recovering said heterodimeric antibody.